Transcriptional Regulation Of Breast Cancer Resistance Protein (BCRP) By P53

Backgroud and Objective: Multidrug resistance(MDR) of cancer is a significant obstacle to successful chemotherapy, which is multifactorial and is known to be mainly conferred by at least three different integral membrane proteins, which are P-glycoprotein(P-gp), multidrug resistanceassociated protein(MRP) and breast cancer resistance protein(BCRP).BCRP is an efflux drug transporter belonging to the subfamily G of the large ABC transporter superfamily. Overexpression of BCRP has been found in numerous types of human cancers and is thought to confer resistance against various clinically relevant compounds, such as mitoxantrone, topotecan, daunorubicin, doxorubicin, methotrexate, SN38 and tyrosine kinase inhibitors(TKIs), etc. To date, little is known about the regulation of BCRP expression, p53 is a critical tumor suppressor, which can be found in 50％of human cancers, and plays an important role in DNA repair, cell cycle arrest and apoptosis, p53 also confers chemosensitivity by regulation of multidrug transporters and other target genes. Up to now, whether p53 regulates BCRP expression has not been studied. In order to further elucidate the mechanisms of MDR, to identify new molecular targets involving in drug resistance conferred by BCRP, and to understand the internal relationship between MDR and tumorigenesis, we investigated the relationship between p53 and BCRP.Methods: p53 overexpression MCF-7 cell lines were established, then the expressions of BCRP and p50 were detected by RT-PCR and Western blot, and the chemosensitivities to mitoxantone(Mit) and 5-fluorouracil (5-Fu) were defined by MTT assay; Endogenous wild type(wt) p53 was disrupted by RNA interference, then the expressions of BCRP and p50 were detected and the phenotype of multidrug resistance was defined. The effects of p53 on the activity of BCRP and NF-κB prompters were detected by Luciferase Reporter Assay. After inhibiting NF-κB activity by chemical inhibitor, MG312 or dominant negative mutant of IκBα, the effect of p53 on the activity of BCRP prompter and the expression of BCRP were detected. Matlnspector program was used to search transcription factor binding sites and related literatures were colleted to screen the transcription factors involved in the regulation of BCRP by p53. EMSA was used to indentify the binding ability of p53 and NF-κB to BCRP promoter;Results: The BCRP and p50 were decreased at the levels of mRNA and protein in wt p53 overexpression MCF-7 cells, but not in the mutant p53 overexpression cells. The RI of wt p53 overexpression MCF-7 cells to Mit decreased 2.76 folds. After silencing the endogenous p53, the expression of BCRP and p50 was increased and the RI of of p53 knockdown cells increased 1.68 folds, wt p53 markedly inhibited BCRP and NF-κB promoter activity in a dose-dependent manner (p＜0.05), while mutant p53 with either mutation in protein-protein interaction domain (p53^Arg175His) or mutation in DNA-binding region (p53^Arg248Trp) of p53 protein lost this inhibitory effect. After NF-κB was inhibited, the relative inhibitory effect of p53 on BCRP promoter activity was lower (p＜0.05) and the expression of BCRP was decreased. Three candidated transcription factors, NF-κB, Sp-1 and NF-Y, were identified by the MatInspector program, p53 could not bind to the BCRP promoter, while NF-κB was able to bind to the NF-κB(p50) binding site within the BCRP promoter.Conclusions: wt p53 may downregulate BCRP expression through a decrease of NF-κB(p50) expression and may enhance the chemosensitivity to some chemotherapeutic drugs. The sites, which invole in the repression of BCRP by wt p53, include 175aa and 248aa. NF-κB may activate BCRP expression through directly binding to the NF-κB(p50) binding site within the BCRP promoter.